Microbial interactions shape cheese flavour formation.

Cheese fermentation and flavour formation are the result of complex biochemical reactions driven by the activity of multiple microorganisms. Here, we studied the roles of microbial interactions in flavour formation in a year-long Cheddar cheese making process, using a commercial starter culture containing Streptococcus thermophilus and Lactococcus strains. By using an experimental strategy whereby certain strains were left out from the starter culture, we show that S. thermophilus has a crucial role in boosting Lactococcus growth and shaping flavour compound profile. Controlled milk fermentations with systematic exclusion of single Lactococcus strains, combined with genomics, genome-scale metabolic modelling, and metatranscriptomics, indicated that S. thermophilus proteolytic activity relieves nitrogen limitation for Lactococcus and boosts de novo nucleotide biosynthesis. While S. thermophilus had large contribution to the flavour profile, Lactococcus cremoris also played a role by limiting diacetyl and acetoin formation, which otherwise results in an off-flavour when in excess. This off-flavour control could be attributed to the metabolic re-routing of citrate by L. cremoris from diacetyl and acetoin towards α-ketoglutarate. Further, closely related Lactococcus lactis strains exhibited different interaction patterns with S. thermophilus, highlighting the significance of strain specificity in cheese making. Our results highlight the crucial roles of competitive and cooperative microbial interactions in shaping cheese flavour profile.

Development of Tyrosine Decarboxylase-Negative Strains of Lactobacillus curvatus by Classical Strain Improvement.

Biogenic amines have been widely studied because of their potential toxicity in fermented foods. Several lactic acid bacteria have the potential to decarboxylate the amino acid tyrosine to tyramine. In this work, we identified two strains of Lactobacillus curvatus, Lbc1 and Lbc2, endowed with the ability to produce tyramine, a metabolic feature that limits their application in starter cultures for fermented meat. To overcome this limitation, we set out to eliminate tyramine production from L. curvatus strains by using classical strain improvement. About 4,000 mutant isolates of both strains were screened using a colorimetric method, and then potential tyrosine decarboxylase-negative mutants were selected. Firm identification of loss-of-function mutants was performed by analytical determination of tyrosine and tyramine in cultivation medium. Of the 8,000 mutants screened, only one mutant of Lbc1 and two mutants of Lbc2 had completely lost the potential to produce tyramine. Subsequently, one tyrosine decarboxylase-negative mutant of both Lbc1 and Lbc2 was characterized in more detail. DNA sequencing of the Lbc1 mutant tdcA gene disclosed two missense mutations in the promoter distal part of the coding sequence. These two mutations result in two amino acid changes in the encoded tyrosine decarboxylase, Pro87Thr and Ser130Leu, presumably inactivating the enzyme activity. The DNA sequence of the other characterized mutant, derived from strain Lbc2, showed that insertion of a 6-bp fragment at nucleotide position 1348 in the tdc gene is presumably the factor leading to loss of activity. With the successful elimination of the undesirable tyramine-producing phenotype without the use of recombinant DNA technology, these developed L. curvatus mutant strains can be safely used in the dairy industry or in the manufacture of various food products.

Biochemical and kinetic characterisation of a novel xylooligosaccharide-upregulated GH43 ß-d-xylosidase/α-l-arabinofuranosidase (BXA43) from the probiotic Bifidobacterium animalis subsp. lactis BB-12.

The Bifidobacterium animalis subsp. lactis BB-12 gene BIF_00092, assigned to encode a ß-d-xylosidase (BXA43) of glycoside hydrolase family 43 (GH43), was cloned with a C-terminal His-tag and expressed in Escherichia coli. BXA43 was purified to homogeneity from the cell lysate and found to be a dual-specificity exo-hydrolase active on para-nitrophenyl-ß-d-xylopyranoside (pNPX), para-nitrophenyl-α-L-arabinofuranoside (pNPA), ß-(1 → 4)-xylopyranosyl oligomers (XOS) of degree of polymerisation (DP) 2-4, and birchwood xylan. A phylogenetic tree of the 92 characterised GH43 enzymes displayed five distinct groups (I - V) showing specificity differences. BXA43 belonged to group IV and had an activity ratio for pNPA:pNPX of 1:25. BXA43 was stable below 40°C and at pH 4.0-8.0 and showed maximum activity at pH 5.5 and 50°C. Km and kcat for pNPX were 15.6 ± 4.2 mM and 60.6 ± 10.8 s-1, respectively, and substrate inhibition became apparent above 18 mM pNPX. Similar kinetic parameters and catalytic efficiency values were reported for ß-d-xylosidase (XynB3) from Geobacillus stearothermophilus T‒6 also belonging to group IV. The activity of BXA43 for xylooligosaccharides increased with the size and was 2.3 and 5.6 fold higher, respectively for xylobiose and xylotetraose compared to pNPX. BXA43 showed clearly metal inhibition for Zn2+ and Ag+, which is different to its close homologues. Multiple sequence alignment and homology modelling indicated that Arg505Tyr506 present in BXA43 are probably important for binding to xylotetraose at subsite +3 and occur only in GH43 from the Bifidobacterium genus.

The long and winding road from the research laboratory to industrial applications of lactic acid bacteria.

Research innovations are constantly occurring in universities, research institutions and industrial research laboratories. These are reported in the scientific literature and presented to the scientific community in various congresses and symposia as well as through direct contacts and collaborations. Conversion of these research results to industrially useful innovations is, however, considerably more complex than generally appreciated. The long and winding road from the research laboratory to industrial applications will be illustrated with two recent examples from Chr. Hansen A/S: the implementation in industrial scale of a new production technology based on respiration by Lactococcus lactis and the introduction to the market of L. lactis strains constructed using recombinant DNA technology.